The present invention relates to a method for secretory production of a human growth hormone having a molecular weight of about 20,000 using Escherichia coli. In particular, the present invention relates to a method for the production of growth hormone having a molecular weight of about 20,000, wherein E. coli is transformed by a recombinant plasmid carrying a DNA fragment in which the 5xe2x80x2 end of a DNA fragment coding for a human growth hormone having a molecular weight of about 20,000 is linked in series to the 3xe2x80x2 end of a DNA fragment coding for E. coli or Salmonella OppA secretion signal, or an amino acid modified form of E. coli OppA secretion signal, the resultant E. coli transformants are cultured, then the resultant culture fluid, cells or processed material thereof are used for the production.
Furthermore, the present invention relates to a method of producing a human growth hormone having a molecular weight of about 20,000, wherein E. coli is transformed with a recombinant plasmid carrying a DNA fragment coding for a human growth hormone having a molecular weight of about 20,000 and a DNA fragment coding for E. coli signal peptidase 1, the resulting E. coli transformants are cultured, then the resulting culture fluid, cells or processed materials thereof are used for the production.
Furthermore, the present invention relates to a method for increasing the production of a human growth hormone having a molecular weight of about 20,000 by altering the medium composition in culturing the transformants transformed by a recombinant plasmid with the abovementioned structure.
There are two known types of human growth hormone (hGH) derived from the pituitary gland: one having a molecular weight of about 22,000 (hereinafter referred to as 22K hGH) and the other having a molecular weight of about 20,000 (hereinafter referred to as 20K hGH).
22K hGH is used for manufacturing pharmaceutical products, by means of recombinant DNA technology, for the treatment of pituitary dwarfism, pediatric chronic renal failure or the like. hGH has recently been found to have excellent activities such as immune promoting activity or lipolysis stimulating activity, as. well as growth promoting activity. Broader applications are greatly expected in the future.
On the other hand, recently, the risks of side effects in the clinical use of 22K hGH are widely reported and have become important issues in securing the safety of GH in clinical use and in broadening its applications. Risks arising from the use of 22K hGH reported to date include possibilities of inducing leukemia and causing diabetes. There is a need for a growth hormone with a lower risk of side effects if its clinical applications are to be broadened.
20K hGH has an amino acid sequence which corresponds to that of 22K hGH consisting of 191 amino acids except that 15 amino acid residues from the 32nd to the 46th inclusive from the N-terminal of 22K hGH are lacked. As compared to 22K hGH, this 20K hGH is reportedly low in leukemia cell proliferation activity and glucose intolerance, which is an index of diabetogenicity. Thus, the risks of side effects reported for 22K hGH are found to be less likely with 20K hGH. Also, according to results of in vitro experiments, 20K hGH has recently been found to be different from 22K hGH in its mode of binding to GH receptors. That is, M. Wada et al. revealed the difference between the two hGHs, showing that the binding activity of 22K hGH to a GH receptor decreases on the cell surface because 22K hGH binds to GH binding protein (GH receptor outer membrane protein) present in the serum at the level of physiological concentration, while a reduction in binding activity of 20K hGH to a GH receptor does not occur because 20K hGH hardly binds to a GH binding protein in physiological conditions, and that in a 1:2 complex (complex of 1 mole of growth hormone and 2 moles of GH receptor), which is necessary for the action of growth hormone via the GH receptor, 22K hGH forms a 1:1 complex (inactive type) while 20K hGH forms a 1:2 complex (active type) with the GH receptor (Mol. Endo 12, 1, 146-156, 19.97). From these results, it is expected that 20K hGH may have higher activity than 22K hGH. It has also been revealed that as to lipolysis stimulating activity, which to date was not clear, 20K hGH is as active as 22K hGH (Japanese Patent Laid-open (Kokai) No. 97/216832, or European Patent Publication No. 0753307).
As mentioned above, 20K hGH has been found to be a growth hormone which exhibits lower risks and higher activity than 22K hGH and has become a novel growth hormone expected to be useful as a pharmaceutical product.
Recent developments in recombinant DNA technology makes it possible to produce heterologous proteins using microorganisms as host organisms. Generally, intracellular expression methods and secretory production methods are used for the production of proteins using microorganisms. In intracellular expression methods, proteins carrying methionine at the N-terminal are accumulated in the cytoplasm. In order to obtain proteins without the methionine residue at the N-terminal, it is necessary to enzymatically cleave amino acid sequences containing the me thionine residue at the N-terminal, for example, by a peptidase. Further, proteins obtained by the intracellular expression methods are not active forms in their steric configurations and require refolding. Thus, intracellular expression methods are not necessarily recommended as efficient methods for protein production.
On the other hand, in secretory production methods using E. coli, target proteins are secreted and accumulated in the periplasm. Purification of proteins from the periplasm extract is easy because the level of impure proteins is lower than that in intracellular expression methods. Moreover, the secreted proteins have no methionine at the N-terminal and are naturally active forms in their steric configuration. Thus, secretory production methods are excellent.
However, secretory production methods using microorganisms require highly sophisticated techniques and thus rarely applied on an industrial scale. That is because precursor proteins synthesized in the cytoplasm pass through the cell membrane and because secretion signals have to be properly cleaved and removed by processing.
As to protein secretion in microorganisms, its mechanisms, relevant factors and their functions have been revealed. Based on these findings, various attempts have been made for efficient secretion methods.
Secretion signals are the first to be discussed. Secretion signals have an extremely important role in protein secretion to lead target proteins to the cytoplasmic membrane. It has been revealed that for the action of secretion signals, a positive charge in the positive charge region of their N terminals and hydrophobicity in the central hydrophobic area are essential (J. Biol. Chem., 267, 4882-4888, 1992). Based on this finding on structural characteristics of secretion signals, Udaka et al. made a modification in which a basic amino acid (Arg) was added to the N terminal positive charge region of a secretion signal of MWP (middle wall protein) derived from Bacillus brevis and a hydrophobic amino acid (Leu) was added to the central hydrophobic region in secretory production using Bacillus brevis as a host, and reported that the secretion efficiency of a sole fish growth hormone was improved to the level of 60 mg/L (Nippon Nogeikagaku Kaishi, 67(3), 372, 1993).
However, there are no theoretical guidelines for the modification of amino acid sequences of secretion signals and at present, preferable sequences have to be found by a try-and-error process. Further, there is no clear principle at present as to how to choose secretion signals derived from secretory proteins for target proteins for secretory production and thus, suitable secretion signals have to be found for the individual target proteins for secretory production.
The abovementioned problems are understood, for example, from the description in Japanese Patent Laid-open (kokai) No. 296491/94. It describes that the secretion signal derived from alkali phosphatase or enterotoxin, which are effective in secretory production of 22K hGH, is not necessarily effective in secretory production of several kinds of eukaryotic proteins. Namely, it reports that there were cases where no protein was synthesized, or proteins were expressed in the cytoplasm but the precursor proteins remained in the cytoplasm and not secreted depending on the combination of secretion signal and secretory protein.
Thus, it is necessary to select secretion signals appropriate to individual secretory proteins; however, to date, no secretion signal usable for secretion of any proteins has been found.
Secondly, the use of co-expression of proteins for the improvement of secretory production is discussed. To date, improvements in secretory production of proteins other than GH were reported, for example, for interleukin 6 by co-expression of SecE and prlA4 (a variant protein of SecY) (BIO/TECHNOLOGY, 12, 178-180, 1994) and human granulocyte-colony stimulating factor (G-CSF) by co-expression of a chaperon protein (Biochem. Biophys. Res. Commun., 210(2), 524-529, 1995).
As to secretory production of 20K hGH, a method in which glutathione reductase is co-expressed with 20K hGH (Japanese Patent Laid-open (Kokai) No. 322586/96; European Patent Publication No. 0735140), and a method in which E. coli Sec protein is co-expressed with 20K hGH (Japanese patent Laid-open (Kokai) No. 313191/97; European Patent Publication No. 0798380) were reported.
The method for the production of 20K hGH by co-expression with glutathione reductase according to the abovementioned Japanese Patent (Kokai) Laid-open No. 322586/96 (or European Patent Publication No. 0735140) enabled the production of about 70 mg per 1 L of culture fluid. However, higher productivity is desired.
Although productivity of secretory production was thus improved by co-expression with glutathione reductase, studies by the present inventors revealed that in this method, a considerable amount of impure proteins other than the target protein, 20K hGH, is contained in the perislasm fluid extract obtained from the cells by an osmotic pressure shock method. As a result, purification of 20K hGH from the periplasm fluid extract comprises many steps, which will result in high production costs to purify 20K hGH to a medically acceptable grade.
An objective of the present invention is to provide a more efficient method for secretory production of 20K hGH using microorganisms, and another objective of the present invention is to provide a method for secretory production of 20K hGH using microorganisms,.in which the level of impure proteins present in the periplasm fluid extract is-decreased.
Objects of the present invention are to provide plasmids containing a DNA fragment in which the 5xe2x80x2 end of DNA encoding 20K hGH links to the 3xe2x80x2 end of DNA encoding a secretion signal which is a modified secretion signal of E. coli OppA, to provide E. coli transformants transformed by said plasmids, and to provide a method for secretory production of 20K hGH using said E. coli transformants.
Other objects of the present invention are to provide a recombinant plasmid having a DNA encoding 20K hGH and a DNA encoding E. coli signal peptidase 1, E. coli transformants transformed with said plasmid, and a method for secretory production of 20K hGH using said E. coli transformants.
To achieve the abovementioned objectives, the present inventors tried to modify a known strain described in Japanese Patent Laid-open (Kokai) No. 322586/96 (or European Patent Publication No. 0735140), i.e., an E. coli transformant, MT-10765 (FERM BP-5020, deposited on Jan. 28, 1995, at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology), which is capable of secretory production of 20K hGH using a modified secretion signal derived from a neutral protease of Bacillus amyloliquefaciens as a secretion signal and glutathione reductase derived from E. coli as a co-expression protein.
First, new secretion signals which could provide more efficient secretory production of 20K hGH were studied.
In order to confirm the effectiveness of new secretion signals, it is necessary to confirm their effect on the amount of secretory production of 20K hGH. The confirmation procedure comprises steps of constructing plasmids each containing a DNA fragment in which the 3xe2x80x2 end of a DNA encoding a different secretion signal is connected to the 5xe2x80x2 terminal of a DNA encoding 20K hGH, isolating E. coli cells transformed by said plasmids, culturing the resulting transformants, then measuring the secretory production of 20K hGH.
The present inventors attempted to increase productivity of 20K hGH by using secretion signals of proteins which are secreted in abundance in microorganisms. However, the inventors could not find secretion signals producing 20K hGH more efficiently by this method than by conventional methods.
Protein secretion is a physicochemical phenomenon in which proteins pass through the cell membrane in an energy-dependent manner. Therefore, the present inventors hypothesized that use of a secretion signal of a protein which is highly homologous to 20K hGH in its structure might be preferable. A protein having an amino acid composition highly homologous to 20K hGH and a protein having a behavior on a purification column similar to 20K hGH were selected as proteins homologous to 20K hGH in their structure. According to a homology search of amino acid sequences, TRBC (Meneewannakul, S. et al., J. Bacteriol., 173, 3872-3878, 1991) and DPPA (Periplasmic Dipeptide Transport Protein Precursor (Dipeptide-binding Protein), Olson, E. R., et al., J. Bacteriol., 173, 234-244, 1991) were selected as proteins highly homologous in their amino acid sequences. E. coli OppA (Periplasmic Oligopeptide Binding Protein, K. Kashiwagi et al., J. Biol. Chem., 265, 8387-8391, 1990) which was revealed by the present inventor to be similar to 20K hGH in its behavior on ion exchange columns and affinity columns was selected as a protein similar to 20K hGH in its behavior on purification columns.
Studies on the effects of secretion signals of the abovementioned-three kinds of proteins on secretory production of 20K hGH showed that the amounts of 20K hGH in secretory production with the secretion signals derived from TRBC and DPPA were less than 1 mg/L, namely there was no increase in secretory production of 20K hGH. Surprisingly, however, it was revealed that the amount of 20K hGH in secretory production using E. coli OppA secretion signal was more than that for MT-10765 (FERM BP-5020) described in Japanese Patent Laid-open (Kokai) No. 322586/96 (or European Patent Publication No. 0735140).
Furthermore, the present- inventors tried to further improve secretory production of 20K hGH by modifying the secretion signal derived from OppA. Results showed that the use of a secretion signal in which an appropriate number-of sequences of the basic amino acid, lysin, were inserted in the positively charged region of the N terminal sequence area and an appropriate number of sequences of the hydrophobic amino acid, leucine, were inserted in the central hydrophobic region attained a secretory production of 20K hGH of more than 110 mg per 1 L culture, namely a productivity higher than that with the use of the secretion signal derived from the natural type of OppA.
It is already known and not included within the scope of present invention that the E. coli OppA secretion signal has an effective role in protein secretion in secretory production in E. coli. However, it is impossible to predict whether the modification of OppA secretion signal would be extremely effective in secretory production of 20K hGH as mentioned above from previously known information.
The present inventors examined the improvement in co-expression of proteins as s secondary trial to achieve the objectives of the present invention.
As a result, the inventors found that the use of signal peptidase 1 derived from E. coli brought secretory production of 20K hGH to a level almost equal to that achieved with the use of glutathione reductase described in Japanese Patent Laid-open (Kokai) No. 322586/96 (or European Patent Publication No. 0735140).
Moreover, surprisingly, it was revealed that the rate of impure proteins other than 20K hGH present in the periplasm solution extracted from cultured cells by an osmotic pressure shock method was extremely low in co-expression with signal peptidase 1 as compared to that in co-expression with glutathione reductase. The reason was not clear but was speculated that signal peptidase 1 strongly suppressed the stress that cells receive by 20K hGH expression, which results in a decreased tendency for lysis of culture cells and thus a reduction in contamination of cytoplasmic proteins into the periplasm extract solution. This reduction of impure proteins in the periplasm solution facilitates the purification of 20K hGH from the periplasm fraction extract solution, and thus markedly contributes to an improvement in purification efficiency and lower costs in manufacturing 20K hGH.
Signal peptidase 1 is an enzyme which cleaves a secretion signal when a precursor protein produced in the cytoplasm passes through the membrane. Jan Maarten van Dijl et al. studied the effects of excessive co-expression of signal peptidase 1 on the secretory production of beta-lactamase when it was connected to various secretion signals (Jan Maarten van Dijl et al., Mol. Gen. Genet., 227, 40-48, 1991). They reported that the effects of co-expression of signal peptidase 1 on the efficiency of processing (cleavage of signals) were different depending on the combination of secretion signal sequences in the precursor and the target protein to be secreted.
However, it is not at all obvious from the abovementioned report whether the co-expression of signal peptidase 1 is effective in improving the efficiency of secretion of 20K hGH, and moreover, it is-not at all known that the tendency of cells to lyse is low such that the amount of impure proteins in the periplasm solution extracted from the cultured cells decreases in co-expression.
Furthermore, the present inventors altered culture conditions to determine a further improvement of secretory production of 20K hGH. As a result, the inventors succeeded in improving the production of 20K hGH up to as high as 82 mg/L by increasing the concentration of carbon source in the culture medium, using a known strain (MT-10765 (FERM BP-5020)) described in Japanese Patent Laid-open (Kokai) No. 32258/96 (also in European Patent Publication No. 0735140).
Accordingly, this method was further applied to MT-10852 (FERM BP-6290) and MT-10853 (FERM BP-6291) (both of which were deposited on Mar. 11, 1998, at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology) of the present invention. As a result, production was successfully increased by 4 to 5 times that of conventional production, i.e., to 350 mg/L using MT-10852 (FERM BP-6290) and to 350 mg/L using MT-10853 (FERM BP-6291), for example, with 3.5% glycerol and 1.5% succinic acid.
Further, the ratio of 20K hGH to total protein was compared, in which the ratio in a periplasm solution extracted from cells of a known strain, MT-10765 (FERM BP-5020), described in Japanese Patent Laid-open (Kokai) No. 322586/96 (or the specification of European Patent Publication No. 0735140) according to the method described in said specification is set to be 1. Results were 3.4 for MT-10853 (FERM BP-6291) in co-expression with glutathione reductase and 8.3 for MT-10852. (FERM BP-6290) in co-expression with signal peptidase 1. Thus, the productivity and purification process for 20K hGH extracted from MT-10852 (FERM BP-6290) and MT-10853 (FERM BP-6291) of the present invention was drastically improved as compared to that of the conventional method, which would enable 20K hGH production costs to be reduced.
The present invention was thus completed.
That is, the present invention comprises:
(1) a secretion signal which is more than 60% homologous to E. coli OppA secretion signal (excluding E. coli OppA secretion signal and Salmonella OppA secretion signal),
(2) a DNA encoding the secretion signal in (1) above,
(3) a recombinant plasmid containing a DNA fragment encoding a secretion signal having more than 60% homology to E. coli OppA secretion signal and a DNA fragment encoding a human growth hormone having a molecular weight of about 20,000, wherein the DNA encoding the human growth hormone having a molecular weight of about 20,000 is connected immediately after the DNA encoding said secretion signal,
(4) a transformant transformed by the recombinant plasmid of (3) above,
(5) a method of producing a human growth hormone having a molecular weight of about 20,000 characterized in that the transformant of (4) above is cultured, a periplasm fraction is extracted from the resulting cells, then the resulting periplasm fraction solution is purified to obtain a human growth hormone having a molecular weight of about 20,000,
(6) A recombinant plasmid containing a DNA fragment encoding a secretion signal, a DNA fragment encoding a human growth hormone having a molecular weight of about 20,000, and a DNA fragment encoding E. coli signal peptidase 1, wherein the 5xe2x80x2 end of the DNA encoding the human growth hormone having a molecular weight of about 20,000 is connected to the 3xe2x80x2 end of the DNA encoding said secretion signal,
(7) a transformant transformed by the recombinant plasmid of (6) above,
(8) a method of producing a human growth hormone having a molecular weight of about 20,000 characterized in that the transformant of (7) above is cultured, a periplasm fraction is extracted from the resulting cells, then the resulting periplasm fraction solution having a small amount of impure proteins is purified to obtain a human growth hormone having a molecular weight of about 20,000.
The present invention also includes a method of increasing production by altering the medium composition in the production of a human growth hormone having a molecular weight of about 20,000 by transformants transformed by the abovementioned recombinant plasmid, namely by using a medium containing at least one of 0.01-10% glycerol and 0.01-5% succinic acid.
The present invention provides a method for secretory production of 20K hGH using E. coli, which enables secretory production of 20K hGH in a larger amount than conventional methods.